CHARACTERIZATION OF SRC KINASE ACTIVATION IN COLON TUMORS.

A RECENT STUDY HAS REPORTED ELEVATED LEVELS OF P60(C-SRC) PROTEIN-TYROSINE KINASE SPECIFIC ACTIVITY IN 100% OF HUMAN COLON CARCINOMA TUMOR TISSUES AND CELL LINES EXAMINED (1). FOUR MECHANISMS COULD ACCOUNT FOR THIS ACTIVATION: I) A POINT MUTATION AT SEVERAL SITES IN THE AMINO TERMINAL HALF OF THE GENOME; II) A SPECIFIC MUTATION AT THE REGULATORY SITE TYR 527; III) A DECREASE IN THE AMOUNT OR SPECIFIC ACTIVITY IN THE SRC TYR 527 SPECIFIC KINASE; OR IV) AN INCREASE IN THE AMOUNT OR SPECIFIC ACTIVITY OF A PHOSPHOTYROSYL PROTEIN PHOSPHATASE SPECIFIC FOR THE SRC TYR 527 SITE. IN PHASE I WE PROPOSE TO CONFIRM THE ACTIVATION OF P60(C-SRC) PROTEIN TYROSINE KINASE ACTIVITY IN HUMAN COLON CARCINOMA CELL LINES BY AUTOPHOSPHORYLATION OF THE IMMUNOPRECIPITATED PROTEIN AND BY DIRECT ASSAY OF THE KINASEACTIVITY IN CELL EXTRACTS, USING EXOGENOUS SUBSTRATES. PHASE I WILL CONCENTRATE ON IDENTIFYING WHICH OF THE ABOVE MECHANISMS IS RESPONSIBLE FOR P60(C-SRC) ACTIVATION IN HUMANTUMOR CELL LINES. IN PHASE II WE PROPOSE TO DEVELOP DIAGNOSTIC REAGENTS FOR EITHER THE ENZYMES WHICH MODULATE P60(C-SRC), OR FOR THE ACTIVATED FORM OF P60(C-SRC) ITSELF. ADDITIONALLY, ASSAYS WILL BE DEVELOPED FOR THERAPEUTIC REAGENTS WHICH INHIBIT P60(C-SRC) ACTIVATION IN COLON CANCERS.